P417: A SINGLE AMINO ACID AT THE PNT DOMAIN OF ERG MEDIATES ITS LEUKEMOGENIC ACTIVITY THROUGH INTERACTION WITH THE NCOR-HDAC3 CO-REPRESSOR COMPLEX.
Authors: E. Kugler, S. Madiwale, D. Yong, Y. Birger, J. A. I. Thoms, D. B. Sykes, M. Yassin, N. Aqaqe, A. Rein, H. Fishman, I. Geron, C.-W. Chen, B. Raught, Q. Liu, M. Milyavsky, J. Pimanda, G. G. Privé, S. Izraeli
Journal: HemaSphere (2022)

Background: The ETS transcription factor ERG is essential for the maintenance of hematopoietic stem cells. However, it has also been implicated as an oncogene in the development of acute leukemia. Our studies and those of others have demonstrated that ERG directly contributes to the initiation and maintenance of lymphoid and myeloid acute leukemia subtypes. Nevertheless, ERG co-factors critically involved in leukemogenesis remain largely uncharacterized. Aims: Here we report a critical role for the conserved amino-acid proline at position 199, at the 3’ end of the PNT domain, for ERG’s leukemogenic activity. Methods: A genomic and proteomic analysis has been used to study the role of P199 in the leukemogenic pathway of ERG. Results: We specifically demonstrate in functional and gene expression assays that P199 is required for ERG-induced myeloid differentiation restriction and for self-renewal and maintenance of murine hematopoietic stem and progenitor cells (HSPC). As P199 is located within the PNT domain (protein interaction domain), we attempted to identify key protein interactions associated with leukemia progression induced by ERG. To this end, we used proximity ligation-mass spectrometry (BioID). In HEK293 cells, proximity maps of WT-ERG and a mutated form of ERG at position 199 (P199L-ERG) were compared. A total of 240 putative protein interactors with a significance analysis of interactome were identified. The hits were highly enriched with chromatin modifiers. Most significantly, the mutation severely impaired ERG’s interaction with components of the NCoR-HDAC3 complex, resulting in a 40% reduction in the number of spectral counts in comparison with wild-type ERG. ChIP sequencing for histone markers conducted on ER-Hoxb8 cells (transformed murine GMPs) demonstrated a decrease in H3K27ac signature at over 1500 unique sites in cells transduced with WT-ERG compared to those transduced with the mutant. Interestingly, these sites were predominantly associated with enhancers of genes expressed in mature myeloid cells. Next, we addressed the role of HDAC3 in ERG’s transcriptional activity. RNA sequencing of human ER-Hoxb8 cells treated with BRD3308 (an HDAC3 inhibitor) for 48 hours was performed. Interestingly, leukemia-associated pathways (Hoxa9-Meis1, CBFA2T3 and repression of myeloid differentiation genes) were ranked at the leading edge of gene sets that were significantly perturbed after HDAC3 inhibition. Moreover, we tested the significance of the ERG/NCoR-HDAC3 interaction in models of human leukemia. We demonstrated that ERG-dependent human AML cells as SKNO1 (AML1-ETO) and TF1 (CBFA2T3-ABDH12) were more sensitive to HDAC3 inhibition (RGFP966) as compared to ERG independent cells. Furthermore, RGFP966 exhibited a significant in-vivo antileukemic effect as measured by the burden of disease in the bone marrow of NSG mice transplanted with SKNO1 cells. By using the CRISPR-dCAS9 system we were able to show that leukemia development is abrogated in mice transplanted with SKNO1 cells expressing a reduced level of HDAC3. Remarkably, this effect was translated into a significant survival advantage. Summary/Conclusion: Taken together, our findings indicate that the aberrant overexpression of ERG maintains HSPCs in an undifferentiated state and promotes AML development. We suggest that the interaction between ERG and the NCoR-HDAC3 complex has an important role in the leukemogenic process, and that HDAC3 inhibition could be beneficial in AML characterized by high ERG expression.